During the last ten years, with an explosion in our knowledge about microbial genomes, biochemical pathways within the cell, metabolic flux analysis, microarray analysis and in silico analysis, industrial microbiology has ventured into manufacturing chemicals from renewable feedstock using biocatalysts.
A 2004 U.S. Department of Energy report, entitled “Top value added chemicals from biomass”, has identified 15 building block chemicals that can be produced from renewable feedstocks using biocatalysts. The 15 building blocks are 1,4-diacids (succinic, fumaric and malic), 2,5-furan dicarboxylic acid, 3-hydroxypropionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, xylitol, and arabinitol. Of these 15 chemicals identified by U.S. Department of Energy, succinic acid production at industrial scale using biocatalysts has advanced significantly (Kurzrock and Weuster-Botz, 2009; Lee et al., 2008; Andersson, et al., 2007; Lu et al., 2009; US Patent Application Publication No. 2006/0073577).
The bacteria residing in the rumen of the cattle are known to produce succinic acid under anaerobic growth conditions. A number of rumen bacteria such as Actinobacillus succinogens, Anaerobiosprillum succiniproducens and Mannheimia succiniproducens have been isolated and developed as biocatalysts for succinic acid production. Escherichia coli strains capable of producing succinic acid in titers, rates, and yields that approach commercial feasibility have also been constructed using the combined knowledge of microbial carbon metabolism and microbial genetics. U.S. Pat. No. 7,223,567 describes construction of E. coli strain SBS550MG producing succinic acid in a rich growth medium. U.S. Pat. No. 6,455,284 describes the construction of an E. coli strain with an exogenous pyruvate carboxylase gene as a biocatalyst for succinic acid production. Pyruvate carboxylase is absent in wild type E. coli strains. The pyruvate carboxylase gene obtained from other microbial organisms such as Rhizobium elti can be expressed under a constitutive promoter to enhance succinic acid production. PCT Patent Application Nos. WO/2008/115958 and WO/2010/115067 describe the construction of E. coli strain KJ122, which is a biocatalyst for succinic acid production in minimal growth medium.
To achieve commercially attractive biosynthetic production of succinic acid and other chemicals, further genetic manipulations are necessary. There is still a need to improve the overall “efficiency” (defined as including, but not limited to, titer, rate, and yield) of production of succinate and other chemicals by microorganisms by means of manipulating the biochemical pathways inside the cell using novel genetic approaches. To the extent that chemical production is coupled to cell growth, an increase in efficiency of succinic acid or other chemical production can also be achieved by means of improving the rate of growth of the microbial cells that produce the desired chemical. The maximum theoretical yield for succinic acid production in E. coli is calculated to be 1.714 mol of succinic acid per 1 mol of glucose providing a mass yield of 1.12 gram of succinic acid for one gram of glucose consumed (Vemuri, et al., 2002).
U.S. Patent Application Publication No. 2008/0009041 describes an E. coli strain comprising chromosomal DNA that is at least 470 kb shorter than that of wild-type E. coli strain. This mutant E. coli strain accumulates more cell mass and exhibits significant increase in the amount of threonine accumulated.
U.S. Patent Application Publication 2009/0075333 also describes an E. coli strain with reduced genome size having one or more of equal or improved growth rate, transformation efficiency, protein expression, DNA production, DNA yield and/or DNA quality compared to the parental strain.
U.S. Patent Application Publication 2009/0221055 describes a novel Bacillus subtilis mutant strain having good productivity of various enzymes derived via gene disruption.
These approaches of reducing the genome size to achieve increased growth rate is based on the fact that the bacterium growing in the fermentor includes many non-essential genes which can be deleted. The bacterium living in a natural environment has many condition-responsive genes to provide mechanisms for surviving difficult environmental conditions of temperature, stress, or lack of food source. Replicating these genes, which are unnecessary in the fermentor, requires expenditure of cellular energy that could be conserved otherwise in the absence of these unnecessary genes.
While the approach of reducing the genome size may be useful for improving the growth performance of the strains producing recombinant proteins, nucleic acid and amino acid, it has not been shown to be a useful approach to improve the performance of strains to produce other chemicals such as succinic acid, fumaric acid, and malic acid, which are intermediates in the Krebs cycle (also known as the tricarboxylic acid cycle or TCA cycle). The efficiency of production of these and other chemicals also depends on the rate of flow of carbon through different paths in the central metabolic pathways and, in some cases, upon achieving a favorable redox balance during anaerobic or microaerobic growth.
The U.S. Patent Application Publication 2009/0075352, and Lee et al (2005) describe a method for improving a bacterial strain based on in-silico analysis. In this approach, the genomic sequence of Mannheimia succiniproducens, which produces succinic acid in significant quantities, is compared with the genomic sequence of E. coli to identify optimal genes to be deleted in E. coli for the purpose of converting a wild type E. coli strain into a succinic acid producing strain. Although this approach looks attractive on the surface, it remains to be seen whether the genetic information derived from M. succiniproducens can be extrapolated to E. coli to achieve a commercially viable succinate production strain. The recommended deletions from the above cited US patent application were a combination of ptsG, pykA, and pykF. However, the succinate titer actually achieved from such a strain was only 8.16 mM (0.96 g/l), which is nowhere near the level needed for commercially attractive production, and growth of the strain was very poor. Moreover, a combination of deletion mutations (pykA, pykF, and ptsHI) similar to that recommended in the application referenced above has been reported earlier, and the resulting strain grew very poorly on glucose (Ponce, et al., 1995). Thus, the combination of mutations described by Lee et al (2005) were not sufficient to construct a commercially viable strain, and the mutations were not tested in a strain context that could prove that they were necessary or appropriate for a commercially viable strain. Therefore, for one skilled in the art, there is not a clear path from the above disclosures to a commercially viable succinate production strain, which will need to grow well on glucose or other inexpensive carbon source and produce at least 20 g/l succinate (170 mM).
In order to compete with petrochemical processes for chemical syntheses, there is a need in the art for developing more efficient biocatalysts for chemical production. The first step is to identify genetic changes in the host chromosome that could contribute to an enhanced chemical production. In view of this circumstance, an object of the present invention is to identify novel mutations that can be used to improve chemical production by microbial strains selected for industrial use. In a specific embodiment, an objective of the present invention is to improve the efficiency of microbial production of organic acids. In a more specific embodiment, the objective is to improve the efficiency of succinic acid production. In another aspect of the invention, the objective is to learn how, in general, to rationally improve production of chemicals such as succinic acid from microbes without having to resort to labor-intensive methods such as metabolic evolution.
A type of reverse engineering has been performed on Corynebacterium glutamicum strains that have been mutagenized and screened for high lysine production. The genome of a highly altered lysine production strain is compared to that of the wild type strain, and the differences were used to re-engineer a minimally mutated strain (Ikeda et al., 2006). However, several hundreds of mutations were found, and although only a few of these seemed to play a major role in lysine overproduction, it would be highly impractical to test all of the several hundred mutations for relevance. On the surface, the invention disclosed herein might seem to be similar to what is disclosed in this prior art, a major difference is that the starting strain of the present invention was not mutagenized, but rather allowed to mutate spontaneously, and it was not screened, but rather selected in a process of metabolic evolution. This results in at least two major differences that distinguish the present invention: (1) the density of mutations is far lower, by almost two orders of magnitude, and (2) all eight of the mutations tested in the present invention proved to be relevant for either improved growth or improved efficiency of chemical production. Thus the methods and materials of the present invention are much improved over those of the prior art.